Name: glutamine flux imaging using genetically encoded sensors
Uploaded: 2014-07-31T13:00:00
Description: Watch this Scientific Journal Video about Glutamine Flux Imaging Using Genetically Encoded Sensors at JoVE.com

This work was supported by NIH grant 1R21NS064412, NSF grant 1052048 and Jeffress Memorial Trust grant J-908.

1. Sample Preparation
Note: In many cases perfusion experiments wash away a significant portion of cells, which can become a frustrating issue. Although not necessary for all cell lines, coating cover glass surfaces with poly-L-Lysine (add 1.0 ml/25 cm2 of 0.01% solution to the surface, incubate &#62;5 min, wash twice with cell culture grade water, and dry in the biosafety cabinet) enhances cell adhesion. Also, be aware of the biosafety level (BSL) of the cell line used, and follow th...

<ol>
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The success of imaging experiments depends upon a few critical factors. One of these factors is the affinity of sensors used, as discussed above. Absolute concentration of the substrate in the subcellular compartment of interest, however, is often unknown. Therefore we recommend trying multiple sensors with staggered affinities to find the one that works best under the desired experimental condition. For example, in our case we transfected the cos7 cells with glutamine sensors with 1.5&#956;, 100&#956;, 2m, and 8m (<stro...

Due to remarkable progress in technologies that allows examination of the transcriptome and the proteome at a cellular level, it has now become clear that the biochemistry and the resulting flux of metabolites and ions are highly cell-type specific. For example, in the mammalian liver, sequential glutamine degradation and synthesis are carried out simultaneously by periportal cells and perivenous cells respectively, feeding ammonium to the urea cycle in the former cell type while consuming excess ammonia in the latter 1-3. In some cases, significant biochemical heterogeneity is detected even in a single &#8220;cell type&#8221; 4,5. In addition to such spatial specificity, the cellular levels of metabolites and ions are highly dynamic (e.g., signaling molecules such as Ca2+ and cyclic nucleotides). The spatiotemporal patterns of metabolites and ions often play critical roles in signal transduction. Monitoring cellular dynamics of metabolites and ions, however, pose unique challenge. In many cases the change in concentrations are rapid and transient, exemplified by the case of signaling molecules such as Ca2+, which decays within ~20 msec in dendritic spines 6. In addition, compartmentalization of biochemical pathways within and among the cells makes it difficult to quantify the dynamics of metabolites and ions using extraction and column chromatography/mass spectrometry techniques.
Genetically encoded sensors for biological molecules are now widely used due to the high spatiotemporal resolution that allows the experimenter to study short-lived and/or compartmentalized molecular dynamics (reviewed in 7,8). These genetically encoded sensors can roughly be divided into two categories; intensity-based sensors and ratiometic sensors. Intensity-based sensors typically consist of a binding domain and a fluorescent protein (FPs), and the solute binding to the binding domain changes the fluorescent intensity. Ratiometic sensors, on the other hand, often take advantage of F&#246;ster Resonance Energy Transfer (FRET) between two FPs that function as a FRET pair. These sensors consist of a binding domain and two FPs, and the solute binding induces the change in FRET efficiency between the two FPs. A large number of sensors for biologically important metabolites and ions have been developed in the past decade 8,9.
One of the exciting possibility offered by such genetically encoded sensors is their use in the high-resolution analysis of membrane transport processes, which previously was not easy to detect at the cellular level. Genetically encoded sensors facilitate the analysis of transport mechanisms such as substrate specificity and pH dependence 10,11. Moreover, in combination with the genetic resources such as the library of RNAi constructs for model organisms, it is now possible to conduct genome-wide searches for novel transport processes using genetically encoded sensors. Indeed, use of genetically encoded sensor lead to the discovery of previously uncharacterized transporters in multiple cases 12,13.
Recently, our laboratory has developed a series of FRET-based sensor for glutamine. We have demonstrated that cellular glutamine levels can be visualized using such FRET glutamine sensors 10. These sensors consist of a FRET donor (mTFP1) inserted into a bacterial glutamine binding protein glnH, and a FRET acceptor (venus) at the C-termini of glnH (Figure 1). FRET efficiency of these sensors decrease upon binding of glutamine, resulting in the decrease of acceptor/donor intensity ratio. Fine regulation of glutamine transport processes is important in biological processes such as neurotransmission 14,15 and the maintenance of urea cycle in the liver 1,16,17.
Here we show the methodology of analyzing transport activities with FRET sensors for glutamine, using a wide-field fluorescence microscope set-up. The goal of experiments shown here are to detect transporter activities in a single cell and to examine substrate specificity of a transiently expressed transporter.

<table border="0" cellpadding="0" cellspacing="0" width="1129">
	<tbody>
		<tr>
			<td>Inverted fluorescent microscope</td>
			<td>Olympus</td>
			<td>IX81F-3-5</td>
			<td>An equivalent inverted fluorescent microscope from other suppliers would also appropriate.</td>
		</tr>
		<tr>
			<td>Exitation filter (mTFP1)</td>
			<td>Omega Optical</td>
			<td>3RD450-460</td>
			<td>band width 450-460 nm</td>
		</tr>
		<tr>
			<td>Exitation filter (Venus)</td>
			<td>Chroma</td>
			<td>ET500/20</td>
			<td>band width 490-510 nm</td>
		</tr>
		<tr>
			<td>Emission filter (mTFP1)</td>
			<td>Chroma</td>
			<td>HQ495/30m</td>
			<td>band width 475-505nm</td>
		</tr>
		<tr>
			<td>Emission filter (Venus)</td>
			<td>Chroma</td>
			<td>ET535/30m</td>
			<td>band width 520-550nm</td>
		</tr>
		<tr>
			<td>Dichroic mirror (FRET channels)</td>
			<td>Chroma</td>
			<td>470dcxr</td>
			<td>long pass, 470nm cut off</td>
		</tr>
		<tr>
			<td>Dichroic mirror (YFP channel)</td>
			<td>Chroma</td>
			<td>89002bs</td>
			<td>passes 445-490nm, 510-560nm, 590-680nm</td>
		</tr>
		<tr>
			<td>mCherry filter set</td>
			<td>Chroma</td>
			<td>49008</td>
			<td>excitation 540-580nm, long pass dichroic with 585nm cut off, emission filter 595-695nm</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Olympus</td>
			<td>U-LH100L-3-5</td>
			<td>LED- or halogen light source that produces stable light intensity, mercury lamps are not recommended</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Qimaging</td>
			<td>Rolera-MGi EMCCD</td>
		</tr>
		<tr>
			<td>apochromatic fluorescence objective</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>perfusion system, ValveBank II</td>
			<td>AutoMate Scientific</td>
			<td>[01-08]</td>
			<td>Other perfusion systems that allow fast solution exchange would also work</td>
		</tr>
		<tr>
			<td>Laminar-flow chambers</td>
			<td>C&#38;L Instruments</td>
			<td>VC-MPC-TW</td>
			<td>Other larminar-flow chambers would also work</td>
		</tr>
		<tr>
			<td>Chambered slide</td>
			<td>Lab-Tek</td>
			<td>154534</td>
			<td>For open-chamber experiments</td>
		</tr>
		<tr>
			<td>perfusion pump</td>
			<td>Thermo Scientific</td>
			<td>74-046-12131</td>
			<td>For open-chamber experiments</td>
		</tr>
		<tr>
			<td>Software supporting ratiometric measurements</td>
			<td>Intellegenent Imaging Innovation</td>
			<td>Slidebook 5.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow biosafety cabinet</td>
			<td>ESCO</td>
			<td>LA-3A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>13-255-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s MEM (DMEM)</td>
			<td>Hyclone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cosmic Calf Serum</td>
			<td>Hyclone</td>
			<td>SH3008703</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum-free medium for transfection (OPTI-MEM I)</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td>Used with Lipofectamine 2000</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution</td>
			<td>sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>25mm circular glass cover slips</td>
			<td>Thermo Scientific</td>
			<td>12-545-102</td>
			<td>in case VC-MPC-TW is used</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668027</td>
			<td>Other transfection reagents can also be used</td>
		</tr>
		<tr>
			<td>Solution A (Hank&#39;s buffer)</td>
			<td>-</td>
			<td>-</td>
			<td>9.7g HANK salt (Sigma H1387), 0.35g NaHCO3, 5.96g HEPES to 1L, pH adjusted to 7.35 with NaOH</td>
		</tr>
		<tr>
			<td>Solution B</td>
			<td></td>
			<td></td>
			<td>Solution A + 0.04mM Gln</td>
		</tr>
		<tr>
			<td>Solution C</td>
			<td></td>
			<td></td>
			<td>Solution A + 0.2mM Gln</td>
		</tr>
		<tr>
			<td>Solution D</td>
			<td></td>
			<td></td>
			<td>Solution A + 1mM Gln</td>
		</tr>
		<tr>
			<td>Solution E</td>
			<td></td>
			<td></td>
			<td>Solution A + 5mM Gln</td>
		</tr>
		<tr>
			<td>Solution F</td>
			<td></td>
			<td></td>
			<td>Solution A + 5mM Ala</td>
		</tr>
	</tbody>
</table>

glutamine flux imaging using genetically encoded sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

Typical time-course experiments are represented in Figure 2. In these experiments, FRET glutamine sensors with affinities of 8 mM (FLIPQTV3.0_8m, Figure&#160;2A and 2B) and 100 &#956;M (FlipQTV3.0_100 &#956; C and D) were co-expressed with an obligatory amino acid exchanger ASCT2 18 in cos7 cells 10. Influx of glutamine is detected as the change in fluorescence intensity ratios between the donor (mTFP1) and the acceptor (venus) (Figure 2A</s...

Watch this Scientific Journal Video about Glutamine Flux Imaging Using Genetically Encoded Sensors at JoVE.com

Glutamine Flux Imaging Using Genetically Encoded Sensors

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

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